Isolation and characterisation of a strain carrying a conditional lethal mutation in the cou gene of Escherichia coli K12.

Summary A strain which carries a mutation conferring clorobiocin resistance and temperature sensitivity for growth was isolated from Escherichia coli K12. Genetic mapping and the molecular weight of the gene product suggest that the mutation is in the cou gene, specifying a sub-unit of DNA gyrase. Nuclear organisation and segregation and placement of septa are grossly abnormal in the mutant at 42°C. RNA synthesis and initiation of DNA replication are also affected at the restrictive temperature but the rate of DNA chain elongation continues almost undisturbed.     

Fungal phytopathogens encode functional homologues of plant rapid alkalinization factor (RALF) peptides

In this article, we describe the presence of genes encoding close homologues of an endogenous plant peptide, rapid alkalinization factor (RALF), within the genomes of 26 species of phytopathogenic fungi. Members of the RALF family are key growth factors in plants, and the sequence of the RALF active region is well conserved between plant and fungal proteins. RALF1‐like sequences were observed in most cases; however, RALF27‐like sequences were present in the Sphaerulina musiva and Septoria populicola genomes. These two species are pathogens of poplar and, interestingly, the closest relative to their respective RALF genes is a poplar RALF27‐like sequence. RALF peptides control cellular expansion during plant development, but were originally defined on the basis of their ability to induce rapid alkalinization in tobacco cell cultures. To test whether the fungal RALF peptides were biologically active in plants, we synthesized RALF peptides corresponding to those encoded by two sequenced genomes of the tomato pathogen Fusarium oxysporum f. sp. lycopersici. One of these peptides inhibited the growth of tomato seedlings and elicited responses in tomato and Nicotiana benthamiana typical of endogenous plant RALF peptides (reactive oxygen species burst, induced alkalinization and mitogen‐activated protein kinase activation). Gene expression analysis confirmed that a RALF‐encoding gene in F. oxysporum f. sp. lycopersici was expressed during infection on tomato. However, a subsequent reverse genetics approach revealed that the RALF peptide was not required by F. oxysporum f. sp. lycopersici for infection on tomato roots. This study has demonstrated the presence of functionally active RALF peptides encoded within phytopathogens that harbour an as yet undetermined role in plant–pathogen interactions.     

A Nonessential Signal Peptidase II (Lsp) of Myxococcus xanthus Might Be Involved in Biosynthesis of the Polyketide Antibiotic TA

Myxococcus xanthus is a gram-negative soil bacterium that produces the polyketide antibiotic TA. In this study, we describe the analysis of an M. xanthus gene which encodes a homologue of the prolipoprotein signal peptidase II (SPase II; lsp). Overexpression of the M. xanthus SPase II in Escherichia coli confers high levels of globomycin resistance, confirming its function as an SPase II. The M. xanthus gene encoding the lsp homologue is nonessential for growth, as determined by specific gene disruption. It has been mapped to the antibiotic TA gene cluster, and the disrupted mutants do not produce the antibiotic, indicating a probable involvement in TA production. These results suggest the existence of more than one SPase II protein in M. xanthus, where one is a system-specific SPase II (for TA biosynthesis).     

Long noncoding RNA H19 in ovarian biology and placenta development

As the first long noncoding RNA to be discovered, H19 has gained substantial attention as a key regulator of several biological processes and its roles in female reproductive biology are gradually getting revealed. Herein, we have summarized the current evidence regarding H19 expression pattern and involvement in the developmental and pathological processes associated with the ovary and the placenta. The findings indicate that within the ovaries, H19 is expressed in the antral and cystic atretic follicles as well as in the corpora lutea but absent in the primordial, primary, and secondary follicles. Its normal expression promotes the maturation of antral follicles and prevents their premature selection for the ovulatory journey while its aberrant induction promotes polycystic ovary syndrome development and ovarian cancer metastasis. In the placenta, H19 is highly expressed in the cytotrophoblasts and extravillous trophoblasts but weakly expressed in the syncytiotrophoblast layer and potentially controls trophoblast cell fate decisions during placenta development. Abnormal expression of H19 is observed in the placental villi of pregnancies affected by pre-eclampsia and fetal growth restriction. Therefore, dysregulated H19 is a candidate biomarker and therapeutic target for the mitigation of ovarian and placenta-associated diseases.     

The Active Component of the Bioemulsifier Alasan from Acinetobacter radioresistens KA53 Is an OmpA-Like Protein

The bioemulsifier of Acinetobacter radioresistens KA53, referred to as alasan, is a high-molecular-weight complex of polysaccharide and protein. Recently, one of the alasan proteins, with an apparent molecular mass of 45 kDa, was purified and shown to constitute most of the emulsifying activity. The N-terminal sequence of the 45-kDa protein showed high homology to an OmpA-like protein from Acinetobacter spp. In the research described here the gene coding for the 45-kDa protein was cloned, sequenced, and expressed in Escherichia coli. Recombinant protein AlnA (35.77 kDa without the leader sequence) had an amino acid sequence homologous to that of E. coli OmpA and contained 70% of the specific (hydrocarbon-in-water) emulsifying activity of the native 45-kDa protein and 2.4 times that of the alasan complex. In addition to their emulsifying activity, both the native 45-kDa protein and the recombinant AlnA were highly effective in solubilizing phenanthrene, ca. 80 microg per mg of protein, corresponding to 15 to 19 molecules of phenanthrene per molecule of protein. E. coli OmpA had no significant emulsifying or phenanthrene-solubilizing activity. The production of a recombinant surface-active protein (emulsification and solubilization of hydrocarbons in water) from a defined gene makes possible for the first time structure-function studies of a bioemulsan.     

Cyanobacterial biomass production in saline media

Summary The cultivation, growth patterns, and physiological activities of the marine cyanobacterium (blue-green alga)Spirulina subsalsa were studied. A comparison of its growth in three different media (diluted seawater, seawater, and seawater +0.5M NaCl) revealed a faster growth in the hypersaline medium. In the hypersaline medium, the culture was homogeneous, in contrast to the aggretates formed in the lower-salt media. Enzymic analysis of the cells demonstrated selective sensitivity of soluble malate dehydrogenase to sodium ions, while chloride ions or nonionic solutes caused no inhibition. The membrane-associated enzyme ferredoxin-NADP reductase was only partially sensitive to sodium ions. The respiratory enzymes exhibited well-coupled activity, and faster respiration was observed with the preparation from the hypersaline culture.     

HOIL‐1 ubiquitin ligase activity targets unbranched glucosaccharides and is required to prevent polyglucosan accumulation

HOIL‐1, a component of the linear ubiquitin chain assembly complex (LUBAC), ubiquitylates serine and threonine residues in proteins by esterification. Here, we report that mice expressing an E3 ligase‐inactive HOIL‐1[C458S] mutant accumulate polyglucosan in brain, heart and other organs, indicating that HOIL‐1’s E3 ligase activity is essential to prevent these toxic polysaccharide deposits from accumulating. We found that HOIL‐1 monoubiquitylates glycogen and α1:4‐linked maltoheptaose in vitro and identify the C6 hydroxyl moiety of glucose as the site of ester‐linked ubiquitylation. The monoubiquitylation of maltoheptaose was accelerated > 100‐fold by the interaction of Met1‐linked or Lys63‐linked ubiquitin oligomers with the RBR domain of HOIL‐1. HOIL‐1 also transferred pre‐formed ubiquitin oligomers to maltoheptaose en bloc, producing polyubiquitylated maltoheptaose in one catalytic step. The Sharpin and HOIP components of LUBAC, but not HOIL‐1, bound to unbranched and infrequently branched glucose polymers in vitro, but not to highly branched mammalian glycogen, suggesting a potential function in targeting HOIL‐1 to unbranched glucosaccharides in cells. We suggest that monoubiquitylation of unbranched glucosaccharides may initiate their removal from cells, preventing precipitation as polyglucosan.